, 2010). Biofilms are organized communities of microorganisms that colonize various biotic surfaces and are embedded in a self-produced matrix (McDougald et al., 2011). Bile was reported to stimulate biofilm formation by some enteric pathogens, for example, Vibrio cholerae and Listeria monocytogenes and the indigenous gut commensal Bacteroides fragilis (Hung et al., 2006; Pumbwe et al., 2007; Begley et al., 2009). Very few studies on biofilm formation by indigenous beneficial gut microbes such as lactobacilli have been published (Lebeer et al., 2007; Kubota et al., 2008). The aim check details of the present study

was to evaluate the use of CRB as a quantitative assay to determine the CSH of 17 probiotic lactobacilli strains from an in-house strain bank collection, characterized in our laboratory (Kruszewska et al., 2002) and grown under normal and gastrointestinal-simulated conditions. Furthermore, the CRB assay of three in-house strains, L. plantarum F44, L. paracasei F8, and L. paracasei F19, and two reference strains, L. rhamnosus GG and the S-layer producing strain L. crispatus

12005, was performed at different pH, ionic strength, with/without cholesterol and with proteolytic enzyme-treated cells on CRB to study the possible role of CRB proteins in CSH. The CRB, CSH and biofilm formation of these five strains grown in the MRS broth supplemented with porcine bile (PB), taurocholic acid (TA) or gastric mucin were evaluated under gastrointestinal-simulated Mirabegron growth conditions. Cholesterol (water soluble), Congo red (CR), crystal violet (CV), proteinase EPZ015666 K, pronase E, taurocholic acid sodium salt (TA) and porcine gastric mucin type III were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) was purchased from VWR International AB (Stockholm, Sweden). All chemicals were of analytical grade. Phenyl methyl sulfphonyl fluoride (PMSF) was purchased from ICN Biomedical (Aurora, OH). De Man Rogosa Sharpe (MRS) agar, blood agar base and Luria–Bertani (LB) agar were purchased from Oxoid Ltd (Basingstoke, UK). Sterile 96-well flat-bottomed polypropylene TPP micro-titre plates were purchased

from Techno Plastic Products AG (Trasadingen, Switzerland). Native PB was pooled from 10 slaughtered pigs, sterilized through a 0.45-μM millipore filter and stored at − 20 °C (Nilsson et al., 2008). The 17 lactobacilli strains analyzed are listed in Table 1. All strains were maintained at − 110 °C in Trypticase soy broth (Oxoid Ltd) with 10% (v/v) glycerol. Frozen cultures were grown on MRS agar and incubated at 37 °C for 48 h. Single colonies were inoculated into 5 mL MRS broth and sub-cultured three times to ensure actively growing cells. A 1-mL aliquot of each culture was inoculated in 10 mL MRS broth and incubated at 37 °C for 24 h. Agar-grown cells were cultured on MRS agar at 37 °C for 48 h. Agar as well as broth-cultured cells were harvested, washed twice with phosphate-buffered saline (PBS, pH 7.